1. Field of the Invention
The present invention relates to a process for producing in plant cells substances useful in agricultural and pharmaceutical fields, by producing large quantities of an exogenous gene or its products in plant cells capable of producing replicase of RNA plant virus, e.g., brome mosaic checks virus (hereinafter referred to as BMV), by genetic engineering techniques. Also, the present invention relates to a process for producing useful transformed plants capable of expressing useful characteristics. The present invention further relates to vectors for plant transformation and vectors capable of producing recombinant RNA as well as transformed plant cells.
2. Description of the Related Art
Development of a method for introducing and expressing an exogenous gene in a plant genome using the Ti plasmid transformation system and of a method for utilizing the multiplication system of a plant virus is under way as a technique for producing useful polypeptides in plant cells or as a method for imparting useful characteristics, for example, plant virus resistance, to plants by useful polypeptide. It is known that in the case of introducing a coat protein gene of tobacco mosaic virus (TMV) into a plant genome using the Ti plasmid transformation system, the amount of coat protein produced is at most 0.01% of the total plant protein (Beachy et al., Annu. Rev. Phytopath. (1990), 28:451-474).
According to this technique, the amount of the product produced by an exogenous gene is dependent on promoter activity which regulates the amount of transcription so that evaluation of promoters capable of imparting a more potent transcription activity becomes necessary. On the other hand, TMV can produce at the maximum 2 g of virus particles per kg of leaves in a host plant.
In the case of a method of utilizing the multiplication system of a plant virus which comprises replacing the exogenous gene of a desired substance for the gene moiety of TMV coat protein and inoculating a host plant with the resulting recombinant, the amount of the desired substance produced was about 1 mg/kg of leaves (Takamatsu et al., EMBO J. (1987), 6:307-311).
Turning to the problem involved in TMV, three kind of genes are overlappingly encoded on one single strand of RNA in TMV. It is thus considered that by replacement of an exogenous gene, the regulating mechanism of TMV replication would be affected. For this reason, use of plant viruses having a virus genome divided on several kinds of single stranded RNAs has been investigated.
As an example, there is a BMV which uses as a host many plants belonging to the family Gramineae and which falls under the bromo virus group. The genome of BMV is composed of three kinds of (+) single stranded RNAs and these RNAs are called RNAs 1, 2 and 3, in terms of decreasingly larger molecular weight. In addition, RNA4 called subgenomic RNA also exists in BMV. These RNAs are enclosed in spherical particles having a diameter of about 26 nm, with RNA1 and 2 being alone, respectively and RNA3 and 4 being together (Lane et al., Adv. Virus Res. (1974), 19:151-220).
The advantages of using BMV are 1) the amount of multiplication in a infected plant cell is high, and 2) its regulating mechanism of virus replication is affected with difficulty on replacement of the exogenous gene in a coat protein gene, and hence, only replicase is required for replication, and 3a protein and coat protein are not concerned with the virus replication, because of the characteristics of the divided genomes. The nucleotide sequence of the entire genome of BMV has already been determined (Ahlquist et al., J. Mol. Biol. (1984), 172:369-383); RNA1 has 3234 bases full length and encodes 1a protein (molecular weight of 109 kilodaltons (KD)), RNA2 has 2865 bases full length and encodes 2a protein (molecular weight of 94 KD), and 1a and 2a proteins are considered to be replicase subunits.
It is thought that in (+)-stranded BMV RNA, (-)-stranded RNA would be synthesized from (+)-stranded RNA in a plant cell by this replicase and using the synthesized (-)-stranded RNA as a template, (+)-stranded RNA would be synthesized in large quantities. On the other hand, RNA3 has 2134 bases full length and encodes the two genetic products of 3a protein (molecular weight of 34 KD), and coat protein (molecular weight of 20 KD) but only the 3a protein encoded on the 5' side is directly translated from RNA3. RNA4 has 876 bases full length, possesses the same sequence as that of the coat protein gene portion of RNA3, and becomes mRNA for coat protein. RNA4 is synthesized from RNA3 in a host cell (Ahlquist et al., J. Mol. Biol. (1981), 153:23-38).
The mechanism shows that (-)-stranded RNA3 is synthesized from (+)-stranded RNA3 and (+)-stranded RNA4 is synthesized from the inside of this (-)-strand (Miller et al., Nature (1985), 313:68-70). Alquist succeeded in expressing chloramphenicol acetyl transferase (CAT) on a high level, by removing most of the coat protein gene from RNA3, introducing CAT gene at the removal site, and infecting bare protoplasts with the resulting recombinant RNA3 together with RNAs 1 and 2. However, they failed to utilize this technique in expression of CAT gene on a plant level (Ahlquist et al., Science (1986), 231:1294-1297).
As described above, in constructing a vector from a virus where the virus genome as represented by BMV, cucumber mosaic virus (hereinafter referred to as CMV) alfalfa mosaic virus (hereinafter referred to as AMV) is divided into 4 RNA chains, BMV has been studied most extensively.
In the method wherein the recombinant RNA3 in which the coat protein gene has been replaced with an exogenous gene is merely mixed with RNAs 1 and 2 and a plant protoplast is inoculated with the mixture to produce the exogenous gene in the protoplast, there is the problem that the amount of expression in each cell is small, because the infection efficiency of the protoplast by the RNA is poor and the recombinant virus RNA cannot be systematically infected.
Furthermore, this technique can not be utilized for obtaining a genetically transformed plant. Moreover, industrial production of virus RNA in vitro has serious disadvantages in view of costs. To overcome these problems, the following method has been developed (Mori et al., J. Gen. Virol. (1992), 73:169-172, U.S. patent application Ser. No. 07/663,164). That is, the method using a genetic engineering technique which comprises constructing genomic RNAcDNA of RNA plant virus including BMV and recombinant cDNA where the coat protein gene of virus genomic RNAcDNA is replaced with an exogenous gene, modifying them to express the virus RNA in a plant cell, and inserting them into the genome of a plant using a plant cell transformation method, such as Ti plasmid, etc., or by a DNA direct introduction method, such as electroporation, etc. Thus virus replicase is produced in all cells and recombinant RNA containing the exogenous gene is replicated to express mRNA of the exogenous gene in large quantities. In this case, multiplication of virus RNA in large amounts causes the plants to be diseased and adversely affects the growth of plants. Therefore, multiplication of virus RNA other than the exogenous gene is not considered to be necessarily required. So, a method for modifying the virus genome to delete the ability of RNAs 1 and 2 to multiply in the case of genomic RNA containing the virus replicase gene, for example, BMV, and as the result, translation of la and 2a protein (BMV replicase) alone, has been developed simultaneously (Mori et al., J. Gen. Virol. (1992), 73:169-172, U.S. patent application Ser. No. 07/663,164).
Further, with BMV as an example, sites of 1a, 2a, 3a and coat protein genes can be considered as replacement sites for exogenous genes, which produce the desired proteins which are not fusion proteins. In case of BMV, depending on the strain, a coat protein of 19 KD (CP2) is known to be also produced in addition to a coat protein of 20 KD (CP1) (Sacher and Ahlquist, J. Virology (1989), 63:4545-4552). As a result of extensive studies on the BMV gene to provide a superior method for production, the present inventors have accomplished this invention.